Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Coffee aroma and flavor are key components in consumer preference for coffee varieties and brands. The characteristic aroma and flavor of coffee stems from a complex series of chemical reactions involving flavor precursors (Maillard reactions) that occur during the roasting of the bean. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, over 800 chemicals and biomolecules have been identified as contributing to coffee flavor and aroma. (Flament, I., 2002 “Coffee Flavor Chemistry” J. Wiley et al. U.K.) Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor in order to meet consumer preferences. Both aroma and flavor may be artificially imparted into coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (flavor). However, to date, there is little information concerning the influence of natural coffee grain components such as polysaccharides, proteins, pigments, and lipids, on coffee aroma and flavor. One approach is to select varieties from the existing germplasm that have superior flavor characteristics. A disadvantage to this approach is that, frequently, the highest quality varieties also possess significant negative agronomics traits, such as poor yield and low resistance to diseases and environmental stresses. It is also possible to select new varieties from breeding trials in which varieties with different industrial and agronomic traits are crossed and their progeny are screened for both high quality and good agronomic performance. However, this latter approach is very time consuming, with one crossing experiment and selection over three growing seasons taking a minimum of 7-8 years. Thus, an alternative approach to enhancing coffee quality would be to use techniques of molecular biology to enhance those elements responsible for the flavor and aroma that are naturally found in the coffee bean, or to add aroma and flavor-enhancing elements that do not naturally occur in coffee beans. Genetic engineering is particularly suited to achieve these ends. For example, coffee proteins from different coffee species may be swapped. In the alternative, the expression of genes encoding naturally occurring coffee proteins that positively contribute to coffee flavor may be enhanced. Conversely, the expression of genes encoding naturally occurring coffee proteins that negatively contribute to coffee flavor may be suppressed.
Coffees from different varieties and origins exhibit significant flavor and aroma quality variations when the green grain samples are roasted and processed in the same manner. The quality differences are a manifestation of chemical and physical variations within the grain samples that result mainly from differences in growing and processing conditions, and also from differences in the genetic background of both the maternal plant and the grain. At the level of chemical composition, at least part of the flavor quality can be associated with variations in the levels of small metabolites, such as sugars, acids, phenolics, and caffeine found associated with grain from different varieties. It is accepted that there are other less well characterized flavor and flavor-precursor molecules. In addition, it is likely that structural variations within the grain also contribute to differences in coffee quality. One approach to finding new components in the coffee grain linked to coffee quality is to study the genes and proteins differentially expressed during the maturation of grain samples in different varieties that possess different quality characteristics. Similarly, genes and proteins that participate in the biosynthesis of flavor and flavor-precursor molecules may be studied.
Carotenoids are one candidate class of flavor and flavor precursor molecules. Carotenoids have been identified in all plants, as well as in a wide range of algae, certain fungi and bacteria (Fraser et al., 1999). Their 40-carbon structure confers particular properties, allowing them to absorb light between about 400 and 500 nm. Carotenoids are liphophilic, a characteristic that, along with their coloration, makes them sensitive to oxidative degradation (Britton, 1988). Carotenoids represent the largest group of pigments in nature, with some 600 different carotenoids identified to date (Cunningham and Grant, 1998). In fact, carotenoid-derived apocarotenoids conceivably constitute one of the largest classes of molecules in nature. Some of the apocarotenoids are essential and valuable constituents of color, flavor, and aroma in edible plants. (Winterhalter and Rouseff, 2002).
In plants, the carotenoid pigments are synthesized in the plastids. The biosynthetic pathway takes place on membranes in the plastid compartment of the cell, and the corresponding genes are located in the nucleus. In chloroplasts, carotenoids accumulate primarily in the photosynthetic membranes in association with the light-harvesting complexes. In the chromoplasts of ripening fruits and flower petals and in the chloroplasts of senescing leaves, the carotenoids may be found in membranes or in oil bodies such as plastoglobules, or in other structures within the stroma.
The first true carotenoid is formed by the condensation of two molecules of geranylgeranyl diphosphate into phytoene (FIG. 1A). This reaction is catalyzed by the enzyme phytoene synthase (geranylgeranyl-diphosphate geranylgeranyl transferase PSY; EC 2.5.1.32). Phytoene, which is not a true pigment since it is unable to absorb light at visible wavelengths, undergoes four consecutive desaturation steps. The first two steps are carried out by the enzyme phytoene desaturase (PDS; EC 1.3.99), resulting in the formation of ζ-carotene (FIG. 1B) via the intermediate phytofluene (Bartley et al., 1992; Hugueney et al., 1992). The second two steps are catalyzed by the enzyme ζ-carotene desaturase (ZDS; EC 1.14.99.30) to form lycopene (FIG. 1C) via the intermediate neurosporene (Albrecht et al., 1995). These desaturation steps require the presence of a plastid terminal oxidase (PTOX) as a co-factor (Carol et al., 1999; Josse et al, 2000; Josse et al., 2003; for review see Kuntz, 2004).
PDS and ZDS yield lycopene (FIG. 1C), the main pigment found in red tomatos. Lycopene serves as the substrate for the formation of both α- and β-carotene via two cyclization reactions. β-carotene (β,β-carotene) (FIG. 1D) is formed by the enzyme lycopene β-cyclase (LβCY; Cunningham et al., 1996), which introduces two β-ring structures at the ends of the carbon chain. This reaction also results in the formation of the intermediate γ-carotene (β,ψ-carotene) containing one β-ring and one uncyclized end, referred to as psi (ψ). Alpha-carotene (β,ε-carotene) (FIG. 1E) is formed by the enzymes lycopene ε-cyclase (LεCY; Ronen et al., 1999) and LβCY, which introduce one ε-ring and one β-ring respectively. The activity of LεCY also results in the formation of the intermediate δ-carotene (ε,ψ-carotene) having one ε-ring and one uncyclized psi end. In plants such as Lactuca sativa (lettuce), LεCY introduces two ε-ring structures at the ends of the carbon chain, resulting in the formation of ε-carotene (ε,ε-carotene; Cunningham and Gantt 2001) (FIG. 1F).
Oxygenated carotenoids are formed by two successive hydroxylation steps. β-carotene (β,β-carotene) is first converted to cryptoxanthine and then zeaxanthine (3,3′-dihydroxy-β,β-carotene) (FIG. 1G) by the action of the enzyme β-carotene hydroxylase (βCHY; EC 1.14.13-; Sandmann., 1994). Alpha-carotene (β,ε-carotene) is also twice hydroxylated; first the β-ring is hydroxylated by βCHY to form the intermediate zienoxanthine (FIG. 1M), and then the ε-ring is hydroxylated by ε-carotene hydroxylase (εCHY) to form lutein (dihydroxy-β,ε-carotene) (FIG. 1H). εCHY has only recently been cloned (Tian at al., 2004; Tian and DellaPenna, 2004; for review see Inoue, 2004), and a lutein deficient mutant (lut1) has been characterized (Pogson et al., 1996; Tian and DellaPenna, 2001).
The hydroxylated β-rings of zeaxanthine are epoxylated in two steps to give antheraxanthine (FIG. 1J) and violaxanthine (FIG. 1K). This reaction is catalyzed by the enzyme zeaxanthine epoxidase (ZEP; Marin et al., 1996; Bouvier et al., 1996). During light stress, violaxanthine can be converted back into antheraxanthine and zeaxanthine due to the activity of violaxanthine de-epoxidase (VDE). ZEP and VDE participate in the xanthophyll cycle, which is implicated in the adaptation of plastids to changing environmental light conditions (for review see Hieber et al., 2000).
Another carotenoid in higher plants is neoxanthine (FIG. 1L). Neoxanthine is synthesized from violaxanthine by a reaction catalyzed by neoxanthine synthase (NYS). NYS was originally cloned from tomato and potato (Bouvier et al., 2000; Al-Babili et al., 2000).
All of the carotenoid substrates described above are available for both oxidative and enxymatic cleavage resulting in the formation of diverse volatile and non-volatile apocarotenoids. Terpenoid flavor volatile compounds are generally present in plants at relatively low levels, but possess strong effects on the overall human appreciation of the flavor, for example, in tomatoes (Buttery et al., 1971 and 1987; Baldwin et al., 1991 and 2000), carrots (Kjeldsen et al., 2003), quince (Lutz and Winterhalter, 1992), and Averrhoa carambola (Winterhalter and Schreier, 1995). Among the more important carotenoid derived volatile compounds are β-ionone, α-ionone, geranylacetone (6,10-dimethyl-5,9-undecadien-2-one), pseudoionone (6,10-dimethyl-3,5,9-undecatrien-2-one) and β-damascenone. Alpha-ionone, β-ionone, β-cyclocitral, and β-damascenone have been shown to contribute approximately 8% of the total aroma intensity and 78% of the total floral aroma category of Valencia orange juice (Mahattanatawee et al., 2005). β-damascenone also contributes to the aroma of grapefruit juice (Lin et al., 2002).
Peak levels of β-ionone and geranylacetone emissions from ripe tomato fruit were calculated to be 1.25 pg/g fw−1 hr−1 and 40 pg/g fw−1 hr−1, respectively. Although β-ionone and geranylacetone are found in low concentrations when compared to other more abundant volatiles such as cis-3-hexenal and hexenal, which have been detected at levels 10,000-fold higher, β-ionone and geranylacetone have odor thresholds of 0.007 mL/L−1 and 60 mL/L−1 respectively (Baldwin et al., 2000). These odor thresholds are significantly lower than that observed for many of the other more abundant volatiles. Thus, carotenoid-derived volatiles have the potential to greatly impact aroma and flavor at low concentrations. β-ionone is considered to be the second most important volatile contributor to tomato fruit flavor (Baldwin et al., 2000).
The biosynthetic routes leading to the formation of apocarotenoid have remained obscure. Based on their chemical structures and studies of volatile production in tomato varieties with unusual carotenoid accumulation, Buttery et al. (1988) predicted that these compounds are likely derived from oxidative carotenoid cleavage. In recent years, a family of carotenoid cleavage dioxygenases (CCDs) that cleave carotenoid substrates at a variety of double bonds have been identified (for review see Bouvier et al., 2005). The first member of the family to be identified was VP14 from Arabidopsis thaliana, a 9-cis-epoxycarotenoid dioxygenase involved in synthesis of xanthoxin (FIG. 1N), the precursor of the phytohormone abscisic acid (ABA; Tan et al., 1997). ABA controls embryo growth potential and endosperm cap weakening during coffee seed germination (da Silva et al., 2004).
Other members of the dioxygenase family, including an Arabidopsis carotenoid cleavage dioxygenase, AtCCD1, that symmetrically cleaves the 9,10(9′,10′) double bonds of multiple carotenoid substrates into a C14 dialdehyde and two C13 cyclohexone derivatives in vitro have been identified (Schwartz et al., 2001). Orthologs of AtCCD1 have been found in a variety of species including Phaseolus vulgaris (Schwartz et al., 2001), Capsicum annuum (Bouvier et al., 2003a), Crocus sativus (Bouvier et al., 2003a) and Petunia hybrida (Simkin et al., 2004a). More recently, Simkin et al. (2004b) demonstrated in transgenic tomato plants that CCD1 enzymes are responsible for the formation of a variety of C13 cyclohexones in vivo. The potential relationships between these volatiles and their carotenoid precursors are shown in FIG. 2. Carotenoid cleaved at the 9,10(9′,10″) bond results in the formation of the corresponding apocarotenoid. Because CCD1 enzymes have 9,10(9′,10″) cleavage specificities, specific products would be generated based on the carotenoid precursor that is present. AtCCD1 and its tomato orthologues are responsible for the formation of geranylacetone and α-ionone (Schwartz et al., 2001; Simkin et al., 2004a and 2004b) and likely β-damascenone (Suzuki et al., 2002). Schwartz et al. (2001) and Simkin et al. (2004b) showed that CCD1s were also capable of forming a number of other important carotenoid-derived volatiles
Schwartz et al. (2001) and Simkin et al. (2004b) purified recombinant AtCCD1 and LeCCD1A enzyme respectively and assayed multiple carotenoid substrates in vitro. The assay products were characterized by thin-layer chromatography and HPLC. In assays containing either β-carotene, zeaxanthine, lutein, violaxanthine and neoxanthine, the central C14 dialdehyde cleavage product (4,9-dimethyldodeca-2,4,6,8,10-pentaene-1,12-dial; I) was the major compound resulting from symmetrical cleavage at the 9,10 and 9′,10′ positions (see FIG. 2). In assays containing β-carotene, zeaxanthine and lycopene, β-ionone (9-apo-β-caroten-9-one; II), 3-hydroxy-β-ionone (3-hydroxy-9-apo-β-caroten-9-one; III) and pseudoionone (V) were formed respectively, whereas α-carotene led to the production of both β-ionone (II) and α-ionone (VI), while δ-carotene led to α-ionone (9-apo-α-caroten-9-one; VI) and pseudoionone (6,10-dimethyl-3,5,9-undecatrien-2-one; V). In assays containing violaxanthine or neoxanthine, 5′6-epoxy-3-hydroxy-β-ionone (5,6-epoxy-3-hydroxy-9-apo-β-caroten-9-one; IV) was formed. Asymmetric cleavage also led to the formation of a C27 epoxy-apocarotenal with these substrates. Several linear carotenoids including phytoene and ζ-carotene are thought to be the precursors of geranylacetone (6,10-dimethyl-5,9-undecatrien-2-one; VII), an important flavor volatile in tomato fruit, and precursors for a second C14 dialdehyde (4,9-dimethyldodeca-4,6,8-triendial; XI).
In assays containing neoxanthine, the asymmetric cleavage also led to the formation of a C27 allenic-apocarotenal and the C13 grasshopper ketone (3,5-dihdroxy-6,7-didehydro-9-apo-β-caroten-9-one; VIII) (see FIG. 3a). The grasshopper ketone is postulated to be the precursor for the formation of β-damascenone (IX) and 3-hydroxy-β-damascenone (X; Suzuki et al., 2002). In assays containing lutein as substrate, symmetrical cleavage at the 9,10 and 9′,10′ positions leads to the formation of both 3-hydroxy-β-ionone (VI) and 3-hydroxy-α-ionone.
Additionally, Bouvier et al (2003b) identified a zeaxanthine-specific 7,8(7′,8′)-cleavage dioxygenase (CsZCD) from Crocus sativus encoding an enzyme capable of forming of crocetin dialdehyde (XII) and 3-hydroxy-β-cyclocitral in vitro (XIII; see FIG. 3b). Crocetin dialdehyde is known to accumulate in the flowers of Jacquinia angustifolia (Eugster et al., 1969) and the roots of Coleus forskohlii (Tandon et al., 1979). 3-hydroxy-β-cyclocitral is believed to be the first committed step in the formation of safranal, a constituent of the spice saffron in C. sativus (Bouvier et al., 2003a). The 7,8(7′,8′)-cleavage of β-carotene by a tomato ZCD orthologue is suspected of being responsible for the formation of β-cyclocitral, contributing to tomato aroma. Bouvier et al. (2003b) have also identified a lycopene-specific 5,6(5′,6′)-cleavage dioxygenase (BoLCD) from Bixa orellana (see FIG. 3c), responsible for the formation of bixin dialdehyde (XIV) and a C7 cleavage product previously identified as 6-methyl-5-hepten-2-one (MHO; XV; Fay et al., 2003). Bixin dialdehyde is the precursor for the formation of the dye bixin/annatto. MHO has been identified as an important contributor to tomato flavor (Buttery et al., 1990; Baldwin et al., 2000).
Despite this extensive knowledge, little work has been done to characterize such volatile molecules in green and roasted coffee. Roasted and un-roasted coffee has been shown to contain two carotenoid derived flavor components, α-ionone and β-damascenone (Czemy et al., 2000; Akiyama et al., 2003; Variyar et al., 2003). The latter component has been identified as a major component of coffee both before and after roasting (Ortiz et al., 2004). These and other carotenoid derived volatile compounds, due to their low odor threshold, require only small amounts to cause a change in aroma.
From the foregoing discussion, it will be appreciated that modulating carotenoid and apocarotenoid content in coffee grain by genetically modulating the production of the proteins responsible for carotenoid and apocarotenoid biosynthesis would be of great utility to enhance the aroma and flavor of coffee beverages and coffee products produced from such genetically engineered coffee beans. Enhanced carotenoid and apocarotenoid content in the coffee bean may also positively contribute to the overall health and wellness of consumers of coffee beverages and products produced from such coffee beans. In addition, modulating carotenoid and apocarotenoid content in the coffee plant has implications for optimizing photosynthesis in conditions of excess or insufficient sunlight. Accordingly, a need exists to identify, isolate and utilize genes and enzymes from coffee that are involved in the biosynthesis of carotenoids and apocarotenoids.